Journal: Cell reports

Article Title: Food-induced dopamine signaling in AgRP neurons promotes feeding

doi: 10.1016/j.celrep.2022.111718

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Goat polyclonal anti-GFP , Rockland Immunochemicals , Cat# 600-101-215, RRID:AB_218182.

Techniques: Plasmid Preparation, Recombinant, Multiplex Assay, Software

Journal: eLife

Article Title: Activity-induced Ca 2+ signaling in perisynaptic Schwann cells of the early postnatal mouse is mediated by P2Y 1 receptors and regulates muscle fatigue

doi: 10.7554/eLife.30839

Figure Lengend Snippet: ( A ) Whole mounts of P7 Wnt1-TdTomato diaphragm were labeled with 488-conjugated α-bungarotoxin (α-BTX). Myelinating Schwann cells along phrenic nerve branches as well as terminal/perisynaptic Schwann cells at α−BTX-labeled neuromuscular junctions (NMJs; green) exhibit tdTomato epifluorescence (red). ( B ) Higher magnification of whole mounts of P7 Wnt1-GCaMP3 diaphragm labeled with GFP, S100, and 633-conjugated α-BTX. All NMJ-associated, S100-immunostained TPSCs express GFP and thus GCaMP3.

Article Snippet: Antibodies against GFP (Rockland), S100 (Dako), synaptophysin (Santa Cruz), neurofilament (Millipore) and acetylcholinesterase (kindly provided by P. Taylor, UCSD) were used at 1/1000 in PBS containing 1% triton-X and 10% fetal bovine serum to detect proteins in fixed, whole-mount diaphragms.

Techniques: Labeling

Journal: eLife

Article Title: Activity-induced Ca 2+ signaling in perisynaptic Schwann cells of the early postnatal mouse is mediated by P2Y 1 receptors and regulates muscle fatigue

doi: 10.7554/eLife.30839

Figure Lengend Snippet: ( A ) Low-power micrographs of confocal images generated from P7 diaphragms of P2ry1 WT (top row) and mutant (bottom row) diaphragm stained with antibodies against GFP (to label GCaMP3-expressing perisynaptic Schwann cells), synaptophysin (Syp; to label presynaptic nerve terminals) and 633-conjugated α-BTX (to label postsynaptic ACh receptor clusters). All NMJs are innervated and display terminal/perisynaptic Schwann cells (TPSCs). ( B ) High-power micrographs of the same tissue also stained with bisbenzamide (Hoechst) exhibit similar patterns of synaptic staining between genotypes. Synaptophysin-immunoreactive presynaptic terminal area, 267 ± 43 vs. 272 ± 47 μm 2 , p=0.85, P2ry1 WT vs. mutant, terminals = 7, n = 3. ( C ) α-BTX-labeled NMJs in both P2ry1 WT (left panels) and mutant (right panels) exhibit robust AChE staining.

Article Snippet: Antibodies against GFP (Rockland), S100 (Dako), synaptophysin (Santa Cruz), neurofilament (Millipore) and acetylcholinesterase (kindly provided by P. Taylor, UCSD) were used at 1/1000 in PBS containing 1% triton-X and 10% fetal bovine serum to detect proteins in fixed, whole-mount diaphragms.

Techniques: Generated, Mutagenesis, Staining, Expressing, Labeling

Journal: The Journal of Neuroscience

Article Title: c-Jun N-Terminal Kinase Phosphorylation of Heterogeneous Nuclear Ribonucleoprotein K Regulates Vertebrate Axon Outgrowth via a Posttranscriptional Mechanism

doi: 10.1523/JNEUROSCI.4821-12.2013

Figure Lengend Snippet: Mutation of the JNK site on hnRNP K has no effect on its subcellular localization. A, B, Nucleocytoplasmic fractionation followed by Western blot analysis of lysates from uninjected WT, EGFP mRNA-, EGFP–hnRNP K mRNA-, S189A mRNA-, and S189D mRNA-injected stage 37/38 embryos. A, Chemiluminescence immunoblot analysis with antibodies to histone H3 (nuclear) and GAPDH (cytoplasmic) indicated complete separation of the fractions. EGFP fusions (EGFP–hnRNP K, S189A, and S189D) and EGFP were all visualized with anti-GFP and compared with endogenous hnRNP K localization. B, Ratios of nuclear versus cytoplasmic band intensities, normalized to histone H3 and GAPDH, respectively, indicated that all EGFP fusions localized similarly to endogenous hnRNP K. Error bars indicate SEM, n = 3 replicates of 40 embryos per group. *p < 0.01, one-way ANOVA with Tukey's post hoc test; ns, nonsignificant (p > 0.9, as before). C–H, Fluorescence immunostaining (magenta) on stage 37/38 hindbrain sections (C, E, G) and primary neuronal cultures (D, F, H). An antibody to nuclear lamins (C, E, G) and DAPI (D, F, H, blue) labeled nuclei, and N-β-tubulin labeled neurons (D, F, H). Arrows (C1–H1) indicate regions measured for intensity profiles (C3–H3). Subcellular localization of EGFP–hnRNP K (C, D), S189A (E, F), and S189D (G, H) was similar to that of endogenous hnRNP K (shown previously in Fig. 3D,E). Scale bars, 20 μm.

Article Snippet: GFP (Rockland) , EGFP , Goat polyclonal , 1:1000 (Western blot) , .

Techniques: Mutagenesis, Fractionation, Western Blot, Injection, Fluorescence, Immunostaining, Labeling

Journal: The Journal of Neuroscience

Article Title: c-Jun N-Terminal Kinase Phosphorylation of Heterogeneous Nuclear Ribonucleoprotein K Regulates Vertebrate Axon Outgrowth via a Posttranscriptional Mechanism

doi: 10.1523/JNEUROSCI.4821-12.2013

Figure Lengend Snippet: The endogenous association of EGFP–hnRNP K with targeted RNAs is unaffected by mutation of the JNK site. A, Experimental design of RNA-binding protein/RNA coimmunoprecipitation to test RNA association in vivo of EGFP fusion proteins (EGFP–hnRNP K, S189A, S189D). After expression of EGFP (control) and EGFP fusions, lysates were incubated with anti-GFP-coated beads to coimmunoprecipitate proteins with bound RNAs. In qRT-PCR, specifically bound RNAs reached threshold before RNAs that were nonspecific and therefore had smaller ΔCT values. B, Real-time qRT-PCR of RNAs eluted from anti-GFP immunoprecipitation, normalized to total input control. ΔCT values of NF-M and tau (hnRNP K targets) were significantly lower for EGFP–hnRNP K, S189A, and S189D compared with that of EGFP control (*p < 0.05, one-way ANOVA with Tukey's post hoc test) but not for peripherin (a nontarget), indicating specific binding. One-way ANOVA further showed no significant (ns) difference in binding of NF-M and tau among EGFP fusion proteins. Error bars indicate SD, n = 3 replicates, 30 embryos per group.

Article Snippet: GFP (Rockland) , EGFP , Goat polyclonal , 1:1000 (Western blot) , .

Techniques: Mutagenesis, RNA Binding Assay, In Vivo, Expressing, Incubation, Quantitative RT-PCR, Immunoprecipitation, Binding Assay

Journal: The Journal of Neuroscience

Article Title: c-Jun N-Terminal Kinase Phosphorylation of Heterogeneous Nuclear Ribonucleoprotein K Regulates Vertebrate Axon Outgrowth via a Posttranscriptional Mechanism

doi: 10.1523/JNEUROSCI.4821-12.2013

Figure Lengend Snippet: Phosphorylation of the JNK site on EGFP–hnRNP K regulates handoff of its RNA targets to the translational machinery. Polysome profiling followed by Western blot analysis of cytosolic extracts from embryos unilaterally coinjected with hnRNP K MO and either EGFP–hnRNP K (A), S189A (B), or S189D mRNA (C). The black line indicates RNA absorbance at 260 nm (A260) across fractions, and premonosomal (Pre), monosomal (Mono), and polysomal (Poly) fractions are indicated (top). Western blots were probed with anti-GFP to visualize the EGFP fusions and anti-S6 to demonstrate proper polysomal separation (bottom). A, EGFP–hnRNP K was most abundant in the premonosomal fractions (94.6%) but was also present to a lesser extent in the monosomal (3.9%) and polysomal fractions (1.5%). B, The loss of phosphorylation at the JNK site prevented S189A from moving beyond the premonosomal fraction (99.7%) into heavier, translating fractions (0.3%). C, S189D, like EGFP–hnRNP K (A), also appeared across all fractions (premonosome, 95.4%; monosome, 3.5%; polysome, 1.1%). D, Quantitation by real-time qRT-PCR of the target (NF-M and tau) and nontarget (peripherin) RNAs of hnRNP K among non-premonosomal fractions (monosome + polysome) with real-time qRT-PCR. Relative amounts (corrected against uninjected WT; see Results) of NF-M (*p = 0.04, one-way ANOVA with Tukey's post hoc test; 3 replicates, 25 embryos per sample) and tau (*p = 0.01, as before) mRNAs in these fractions for embryos unilaterally coinjected with hnRNP K MO and S189A RNA were significantly reduced compared with those coinjected with EGFP–hnRNP K or S189D RNA, whereas relative amounts of peripherin mRNA were not significantly different among groups (p = 0.2, one-way ANOVA as above). Error bars indicate pooled SDs. ns, Nonsignificant.

Article Snippet: GFP (Rockland) , EGFP , Goat polyclonal , 1:1000 (Western blot) , .

Techniques: Western Blot, Quantitation Assay, Quantitative RT-PCR